Metabolic engineering of Escherichia coli for high-yield uridine production.

Uridine is a kind of pyrimidine nucleoside that has been widely applied in the pharmaceutical industry. Although microbial fermentation is a promising method for industrial production of uridine, an efficient microbial cell factory is still lacking. In this study, we constructed a metabolically engineered Escherichia coli capable of high-yield uridine production. First, we developed a CRISPR/Cas9-mediated chromosomal integration strategy to integrate large DNA into the E. coli chromosome, and a 9.7 kb DNA fragment including eight genes in the pyrimidine operon of Bacillus subtilis F126 was integrated into the yghX locus of E. coli W3110. The resultant strain produced 3.3 g/L uridine and 4.5 g/L uracil in shake flask culture for 32 h. Subsequently, five genes involved in uridine catabolism were knocked out, and the uridine titer increased to 7.8 g/L. As carbamyl phosphate, aspartate, and 5'-phosphoribosyl pyrophosphate are important precursors for uridine synthesis, we further modified several metabolism-related genes and synergistically improved the supply of these precursors, leading to a 76.9% increase in uridine production. Finally, nupC and nupG encoding nucleoside transport proteins were deleted, and the extracellular uridine accumulation increased to 14.5 g/L. After 64 h of fed-batch fermentation, the final engineered strain UR6 produced 70.3 g/L uridine with a yield and productivity of 0.259 g/g glucose and 1.1 g/L/h, respectively. To the best of our knowledge, this is the highest uridine titer and productivity ever reported for the fermentative production of uridine.

Metabolic engineering of Bacillus subtilis for the co-production of uridine and acetoin.

In this study, a uridine and acetoin co-production pathway was designed and engineered in Bacillus subtilis for the first time. A positive correlation between acetoin and uridine production was observed and investigated. By disrupting acetoin reductase/2,3-butanediol dehydrogenasegenebdhA, the acetoin and uridine yield was increased while 2,3-butanediol formation was markedly reduced. Subsequent overexpression of the alsSD operon further improved acetoin yield and abolished acetate formation. After optimization of fermentation medium, key supplementation strategies of yeast extract and soybean meal hydrolysate were identified and applied to improve the co-production of uridine and acetoin. With a consumption of 290.33 g/L glycerol, the recombinant strain can accumulate 40.62 g/L uridine and 60.48 g/L acetoin during 48 h of fed-batch fermentation. The results indicate that simultaneous production of uridine and acetoin is an efficient strategy for balancing the carbon metabolism in engineered Bacillus subtilis. More importantly, co-production of value-added products is a possible way to improve the economics of uridine fermentation.

Benefits of heat-killed Lactobacillus acidophilus on growth performance, nutrient digestibility, antioxidant status, immunity, and cecal microbiota of rabbits.

Introduction: Heat-killed probiotics, as a type of inactivated beneficial microorganisms, possess an extended shelf life and broader adaptability compared to their live counterparts. This study aimed to investigate the impact of heat-killed Lactobacillus acidophilus (L. acidophilus, LA) - a deactivated probiotic on the growth performance, digestibility, antioxidant status, immunity and cecal microbiota of rabbits. Methods: Two hundred weaned Hyla rabbits were randomly allocated into five equal groups (CON, L200, L400, L600, and L800). Over a 28-day period, the rabbits were fed basal diets supplemented with 0, 200, 400, 600, and 800 mg/kg of heat-killed LA, respectively. Results: Results revealed a significant reduction in the feed-to-gain ratio (F/G) in the L600 and L800 groups (p < 0.05). Additionally, the L800 group exhibited significantly higher apparent digestibility of crude fiber (CF) and crude protein (CP) (p < 0.05). Regarding digestive enzyme activities, enhanced trypsin and fibrinase activities were observed in the L600 and L800 groups (p < 0.05). Concerning the regulation of the body's antioxidant status, the L800 group demonstrated elevated levels of superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in both serum and ileal tissue (p < 0.05). In terms of immune capacity modulation, serum tumor necrosis factor-α (TNF-α) levels were significantly lower in the L600 and L800 groups (p < 0.05), while immunoglobulin A (IgA) and immunoglobulin M (IgM) levels were higher (p < 0.05). Additionally, the L800 group exhibited a substantial increase in secretory immunoglobulin A (SIgA) levels in the intestinal mucosa (p < 0.05). In comparison to the CON group, the L800 group exhibited a significant increase in the relative abundance of Phascolarctobacterium and Alistipes in the cecum (p < 0.05). Phascolarctobacterium demonstrated a positive correlation with SIgA (p < 0.05), IgM (p < 0.01), and Glutathione peroxidase (GSH-Px) (p < 0.05), while displaying a negative correlation with TNF-α levels (p < 0.05). Concurrently, Alistipes exhibited positive correlations with IgA (p < 0.05), IgM (p < 0.05), SIgA (p < 0.01), GSH-Px (p < 0.05), SOD (p < 0.05), and T-AOC (p < 0.01), and a negative correlation with TNF-α (p < 0.05). Discussion: In conclusion, the dietary incorporation of 600 mg/kg and 800 mg/kg of heat-killed LA positively influenced the growth performance, nutrient digestibility, antioxidant status, immune capacity and cecal microbiota of rabbits. This highlights the potential benefits of utilizing heat-killed probiotics in animal nutrition.